Loss of RPGR disrupts motile cilia and causes primary ciliary dyskinesia by affecting F-actin dynamics
Yang Wu, Erika Tavares, Binrun Liang, Wallace B. Wee, Vito Mennella, Han-Chao Feng, Jiaying Cao, Pui Yee Wong, Jiayi Zheng, Mu He, Kirk AJ Stephenson, Liran Hanan Hochma, Janice Min Li, Nan-Peng Chen, Sharon D. Dell, Elise Heon, Zhen Liu
Yang Wu, Erika Tavares, Binrun Liang, Wallace B. Wee, Vito Mennella, Han-Chao Feng, Jiaying Cao, Pui Yee Wong, Jiayi Zheng, Mu He, Kirk AJ Stephenson, Liran Hanan Hochma, Janice Min Li, Nan-Peng Chen, Sharon D. Dell, Elise Heon, Zhen Liu
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Research Article Cell biology Pulmonology

Loss of RPGR disrupts motile cilia and causes primary ciliary dyskinesia by affecting F-actin dynamics

Abstract

Cilia are cellular organelles that extrude from the surface of various cell types, serving either sensory or motile functions. Retinitis pigmentosa GTPase regulator (RPGR) variants affect both photoreceptor sensory cilia and airway motile cilia, leading to retinitis pigmentosa (RP) and primary ciliary dyskinesia (PCD), respectively. Not all patients develop PCD, and it remains unclear which RPGR variants predispose patients to PCD. Here, we leverage 2D organoids, super-resolution microscopy, and live-cell imaging to characterize the multiciliated cells (MCCs) from patients with different RPGR variants and CRISPR-modified RPGR KO MCCs. We demonstrate that MCCs with RPGR variants have reduced ciliation, shorter cilia, impaired cilia beat, or cilia beat incoordination, potentially resulting in compromised mucociliary clearance and lung diseases. Moreover, we show that RPGR regulates motile cilia through interfering with F-actin dynamics, evidenced by the undissolved F-actin meshwork in RPGR-deficient MCCs, and the defects can be ameliorated with either latrunculin A or Y27632 treatment. Though PCD was observed only in patients with variants that affect both isoforms, patients with RPGRORF15 variants also had cilia and airway anomalies. All RPGR variants affected motile cilia in some way, and the mechanisms involved the accumulation of apical F-actin.

Authors

Yang Wu, Erika Tavares, Binrun Liang, Wallace B. Wee, Vito Mennella, Han-Chao Feng, Jiaying Cao, Pui Yee Wong, Jiayi Zheng, Mu He, Kirk AJ Stephenson, Liran Hanan Hochma, Janice Min Li, Nan-Peng Chen, Sharon D. Dell, Elise Heon, Zhen Liu

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Figure 8

LatA and Y27632 treatment ameliorated the motile cilia defect in patient MCCs with RPGR variants.

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LatA and Y27632 treatment ameliorated the motile cilia defect in patient...
(A) Immunostaining showed that LatA and Y27632 can partially rescue cilia length and ciliation defects in the MCCs from a patient with RP (patient 9). Scale bar: 10 μm. (B and C) The cilia length and ciliation level in MCCs from patients with pathological variants (patients 9 and 18) were improved by LatA and Y27632 treatment. (D and E) The distribution of Vangl1 and gelsolin in patient cells with RPGR defects can be rescued by Y27632 but not LatA. Scale bar: 10 μm. (F–H) The cilia beat anomalies in MCCs from patients with RPGRex1–19 or RPGRORF15 pathological variants were alleviated by either LatA or Y27632 treatment. (F) Both treatments improved cilia beat frequency (CBF). (G) Both treatments partially rescued the waveform, with more cells displaying a normal beating pattern. (H) The disrupted cilia beat coordination was partially rescued by LatA or Y27632 treatment. (B, C, F–H) Data represent more than 11 of 17 patients with RPGRex1–19 or RPGRORF15 variants. The detailed summary information can be found in Supplemental Figure 17. Data represent mean ± SEM. The center, upper, and lower lines represent the median, upper, and lower quartiles, respectively (F and H). **P < 0.01, ****P < 0.0001 by 2-tailed t test (B, C, F, and H) or Fisher’s exact test (G). (I) A summary cartoon shows that the cilia defects in RPGR KO MCCs or RPGR-deficient patient MCCs can be alleviated by Y27632 treatments.